Chemical-genetic interrogation of small molecule mechanism of action in S. cerevisiae
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Date
24/11/2011Author
Spitzer, Michaela
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Abstract
The budding yeast S. cerevisiae is widely used as a model organism to study
biological processes that are conserved among eukaryotes. Di fferent genomic approaches
have been applied successfully to interrogate the mode of action of small
molecules and their combinations. In this thesis, these technologies were applied
to di fferent sets of chemical compounds in the context of two collaborative
projects. In addition to insight into the mode of action of these molecules, novel
approaches for analysis of chemical-genetic pro files to integrate GO annotation,
genetic interactions and protein complex data have been developed.
The fi rst project was motivated by a pressing need to design novel therapeutic
strategies to combat infections caused by opportunistic fungal pathogens. Systematic
screens of 1180 FDA approved drugs identifi ed 148 small molecules that
exhibit synergy in combination with
uconcazole, a widely used anti-fungal drug
(Wright lab, McMaster University, Canada). Genome-wide chemical-genetic profiles for 6 of these drugs revealed two di fferent modes of action of synergy. Five of
the compounds a ffected membrane integrity; these chemical-genetic interactions
were supported by microscopy analysis and sorbitol rescue assays. The sixth
compound targets a distinct membrane-associated pathway, sphingolipid biosynthesis.
These results not only give insight into the mechanism of the synergistic
interactions, they also provide starting points for the prediction of synergistic
anti-fungal combinations with potential clinical applications. The second project characterised compounds that aff ected melanocytes in a
chemical screen in zebra fish (Patton lab, Edinburgh). Chemical-genetic screens
in S.cerevisiae enabled us to show that melanocyte pigmentation reducing compounds
do so by interfering with copper metabolism. Further, we found that
defects in intracellular AP1 and AP3 trafficking pathways cause sensitivity to low
copper conditions. Surprisingly, we observed that the widely-used MAP-kinase
inhibitor U0126 a ffects copper metabolism. A nitrofuran compound was found to
speci fically promote melanocyte cell death in zebrafi sh. This enabled us to study
off -target eff ects of these compounds that are used to treat trypanosome infections.
Nifurtimox is a nitrofuran prodrug that is activated by pathogen-specifi c
nitroreductases. Using yeast and zebra fish we were able to show that nitrofurans
are also bioactivated by host-specifi c aldehyde dehydrogenases suggesting that a
combination therapy with an aldehyde dehydrogenase inhibitor might reduce side
e ffects associated with nifurtimox.
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